Sulfonamide resistance genes and their use

ABSTRACT

Plant cells are transformed by: 
     (i) transforming a plant cell whose growth is sensitive to inhibition by a sulfonamide or a salt thereof with a chimaeric gene comprising (a) a plant promoter, (b) a sulfonamide resistance gene having a sequence encoding a transit peptide fused to the 5&#39;-end of the resistance gene and (c) a plant polyadenylation/terminator sequence; 
     (ii) selecting a transformed plant cell whose growth is resistant to inhibition by a sulfonamide or salt thereof; 
     (iii) optionally, regenerating from the transformed plant cell a genetically transformed plant which exhibit the said resistance; 
     (iv) optionally, obtaining seed from the regenerated plant; and 
     (v) optionally, propagating plants from the seed. 
     The growth of weeds can be controlled by a locus where a transgenic plant obtained as above is being cultivated by applying to the locus an effective amount of a herbicide, such as asulam, which acts by inhibiting dihydropteroate synthase. The sulfonamide resistance gene can be also used as a selectable marker. The sequence of a sulfonamide resistance gene is presented.

This application is a continuation of application Ser. No. 08/102,395,filed Aug. 5, 1993, which is a continuation of Ser. No. 07/985,352 filedDec. 1, 1992, which is a continuation of Ser. No. 07/746,306, filed Aug.14, 1991, which is a continuation of Ser. No. 07/429,227, filed Oct. 31,1989, all abandoned.

This invention relates to sulfonamide resistance genes.

Sulfonamides are anti-bacterial compounds which act as inhibitors ofdihydropteroate synthase (DHPS), an enzyme of the folic acid synthesispathway. Resistance to sulfonamides is conferred on bacteria by variousR plasmids in Enterobacteriaceae and is found in transposons such asTn21 (De La Cruz and Grinsted. J. Bact. 151, 1982, 222-228) and Tn2603(Yamamoto et al, Mol. Gen. Genet. 181, 1981, 464-469). A sulfonamideresistance gene (sul) codes for a modified DHPS which is insensitive toinhibition by sulfonamides.

According to the present invention, there is provided a method oftransforming a cell, which method comprises

(i) transforming a plant cell whose growth is sensitive to inhibition bya sulfonamide or a salt thereof with a chimaeric gene comprising (a) aplant promoter, (b) a sulfonamide resistance gene having a sequenceencoding a transit peptide fused to the 5'-end of the resistance geneand (c) a plant polyadenylation/terminator sequence;

(ii) selecting a transformed plant cell whose growth is resistant toinhibition by a sulfonamide or salt thereof;

(iii) optionally, regenerating from the transformed plant cell agenetically transformed plant which exhibit the said resistance;

(iv) optionally, obtaining seed from the regenerated plant; and

(v) optionally, propagating plants from the seed.

Transformed cells can be selected whose growth is resistant to apredetermined concentration of a sulfonamide, such as asulam, or a saltthereof. Plants which are resistant to asulam and its salts can beregenerated from transformed cells. Asulam is methyl(4-aminobenzenesulphonyl)-carbamate. That resistance of cells orregenerants is due to the integration and expression of the sul gene maybe verified by assaying for the presence of sulfonamide-insensitiveDHPS.

A DNA fragment suitable for use in the method of the invention comprisesa sulfonamide resistance gene having a sequence encoding a transitpeptide fused to the 5'-end of the resistance gene. The invention alsoprovides a vector which comprises a chimaeric gene comprising (a) aplant promoter; (b) a sulfonamide resistance gene having a sequenceencoding a transit peptide fused to the 5'-end of the resistance geneand (c) a plant polyadenylation/terminator sequence, such that the geneis capable of being expressed in a plant cell transformed with thevector. Plant cells are provided which are transformed with thechimaeric gene.

A transgenic plant according to the invention contains in its cells achimaeric gene comprising (a) a plant promoter, (b) a sulfonamideresistance gene having a sequence encoding a transit peptide fused tothe 5'-end of the resistance gene and (c) a plantpolyadenylation/terminator sequence, such that the plant exhibitsresistance to a herbicide which acts by inhibiting dihydropteroatesynthase. Seed can be obtained from the transgenic plant.

The invention also can be used to kill selectively weeds. The inventionprovides a method of controlling the growth of weeds at a locus, whichmethod comprises applying an effective amount of a herbicide which actsby inhibiting dihydropteroate synthase to a locus where a transgenicplant of the invention is being cultivated. The herbicide is preferablyasulam or an agriculturally acceptable salt thereof.

A sulfonamide resistance gene can also be used as a selectable marker,for example on the basis of the resistance it confers to sulfadiazine,asulam and their salts. A gene encoding another trait of interest can belinked, for example fused, to a sulfonamide resistance gene andintroduced into cells. The cells are then grown on a medium containing asulfonamide or one of its salts. Cells which survive can be assumed tohave been transformed not only with the sulfonamide resistance gene butwith the other gene as well.

Accordingly, the present invention provides a DNA fragment comprising asulfonamide resistance and, linked thereto, a gene encoding a secondtrait such that, when the fragment is used to transform a cell, theexpression of sulfonamide resistance by the cell can be utilised as anindicator that the cell has been transformed by both the sulfonamideresistance gene and the gene coding for the second trait. The inventionadditionally provides a method of transforming a cell, which methodcomprises:

(i) co-introducing a sulfonamide resistance gene and a gene encoding asecond trait into a cell whose growth is sensitive to inhibition by asulfonamide or a salt thereof; and

(ii) selecting a transformed cell whose growth is resistant toinhibition by a sulfonamide or salt thereof. Such a selected cell canthen be screened for the presence of the second gene.

The invention further provides a new sulfonamide resistance gene. Thisgene consists of DNA encoding a mutated DHPS of the sequence: ##STR1##optionally modified by one or more amino acid insertions and/ordeletions and/or by an extension at either or both ends provided thatresistance to asulam is conferred on a cell when a gene encoding such amodified sequence is expressed therein.

A preferred gene has the sequence: ##STR2## optionally modified by oneor more codon insertions and/or deletions and/or by an extension ateither or each end provided that resistance to a sulfonamide isconferred on a cell when the modified gene is expressed therein.

A modified gene sequence may be obtained by introducing correspondingchanges into the DNA sequence encoding the unmodified amino acidsequence of the sul gene of the invention. This may be achieved by anyappropriate technique, including restriction of the DNA sequence with anendonuclease, insertion of linkers, use of an exonuclease and/or apolymerase and site-directed mutagenesis.

A shorter DNA sequence therefore may be obtained by removing nucleotidesfrom the 5'-terminus or the 3'-terminus of the DNA sequence encoding theunmodified amino acid sequence, for example using an exonuclease such asBal 31. Whether a modified DNA sequence encodes a modified proteincapable of conferring resistance to asulam may be readily ascertained.The modified DNA sequence can be cloned into an appropriate plasmid anda host cell transformed and tested for sulfonamide resistance, forexample resistance to asulam or sulfadiazine.

As far as extensions are concerned, the amino acid sequence of the sulgene may be extended by up to 70 amino acids at either or both ends. Upto 50 amino acids, for example up to 20 amino acids, may therefore beadded to the N-terminus and/or the C-terminus. Such an extension maytherefore be, at the N-terminus, a transit peptide sequence. A transitpeptide sequence is required when transforming plant cells with the aimof generating plants resistant to herbicides which act by inhibitingdihydropteroate synthase.

Where the sequence of the mutated DHPS encoded by the gene according tothe invention is modified, typically there is a degree of homology of atleast 70% between the modified and unmodified sequences. For example,the degree of homology may be 85% or more or 90% or more. In any eventresistance to asulam, including to an agriculturally acceptable saltthereof, must be conferred on a cell when a gene encoding a modifiedsequence is expressed in the cell.

For the purposes of amplification, manipulation and transformation, asulfonamide resistance gene is typically part of a larger DNA fragment.Such larger fragments may be up to 2.0 kb, for example up to 1.25 kb.Such a fragment is typically provided with restriction sits are bothends to facilitate manipulation. It preferably incorporatestranscriptional regulatory sequences and/or, if not present at the3'-end of the coding sequence of the gene, a stop codon for thesulfonamide gene. A fragment may therefore also incorporate a promoter,Shine-Delgarno sequence and terminator sequence which are capable ofenabling the sul gene to be expressed in plant cells or in whicheverother type of cells are to be transformed by the gene.

The fragment may be a hybrid fragment in which the sul gene promoter hasbeen replaced by another promoter. The promoter may be a plant promoter,for example the 35S cauliflower mossic virus promoter or a nopalinesynthase or octopine synthase promoter. Where manipulations in bacteriaare undertaken, a bacterial promoter such as the lac promoter may bepresent.

In one aspect of the invention, plant cells are transformed with achimaeric sul gene comprising (a) a plant promoter operably linked to(b) a sul gene having a sequence encoding a transit peptide fused to the5'-end of the said sul gene, and (c) a plant polyadenylation/terminatorsequence. Plant cells can be transformed with the chimaeric genedirectly, typically by way of a DNA fragment comprising the chimaericgene. Alternatively, there may be used a vector incorporating thechimaeric gene. The chimaeric gene includes transcriptional controlsequences, for example as above, and translational initation and/ortermination sequences. A vector typically contains too a region whichenables the chimaeric gene to be transferred to and stably integrated inthe plant cell genome.

Any sul gene may be employed, although the gene is typically a bacterialgene. The sul gene may be a sulI or sulII gene. A sul gene according tothe invention may be employed, although optionally there may be basechanges which do or do not result in amino acid substitutions in theexpressed product. The sul gene is provided with a transit peptidesequence at its 5'-end. The encoded transit peptide is able to targetthe modified DHPS encoded by the sul gene into the chloroplast stroma.The chimaeric gene comprising a sul gene having a sequence encoding atransit peptide fused to the 5'-end of the resistance gene is typicallyprepared by ligating the transit peptide sequence to the 5'-end of theresistance gene. The transit peptide sequence may be the transit peptidefor ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO). The peaRUBISCO transit peptide may be employed.

Preferably the sul coding sequence is fused to a transit peptide codingsequence in such a way that cleavage between the transit peptide and themodified DHPS encoded by the sul gene releases the mature modified DHPSinto the chloroplast stroma. In other words, the sul gene is not fusedto codons encoding the N-terminal amino acid residues of the matureprotein which is normally targetted to the chloroplast stroma by thetransit peptide. This is in contrast to the targetting efficiency of afusion between the RUBISCO transit peptide and 5-enolpyruval shikimatephosphate (EPSP). Such a fusion protein is not targetted to thechloroplast stroma when the N-terminal sequence of the mature RUBISCOprotein is not present (Comai et al, J. Biol. Chem. 263, 15104-15109,1988).

Transformed plant cells can be selected by growth in a medium containinga sulfonamide or a salt thereof, such as sulfadiazine, asulam or anagriculturally acceptable salt thereof. Plant tissue can therefore beobtained comprising a plant cell which incorporates a sul gene in theplant cell genome, the gene being expressible in the plant cell. Plantscan then be regenerated which include the sul gene in the plant cellgenome such that the gene can be expressed. The regenerated plants canbe reproduced by conventional means and, for example, seed obtained.

A preferred way of transforming a plant cell is to use Agrobacteriumtumefaciens containing a vector comprising a chimaeric sul gene asabove. A hybrid plasmid vector may therefore be employed whichcomprises:

(a) the chimaeric gene under the control of regulatory elements capableof enabling the gene to be expressed when integrated in the genome of aplant cell;

(b) at least one DNA sequence which delineates the DNA to be integratedinto the plant genome; and

(c) a DNA sequence which enables this DNA to be transferred to the plantgenome.

Typically the DNA to be integrated into the plant cell genome isdelineated by the T-DNA border sequences of a Ti-plasmid. If only oneborder sequence is present, it must be the right border sequence. TheDNA sequence which enables the DNA to be transferred to the plant cellgenome is generally the virulence (vir) region of a Ti-plasmid.

The sul gene and its transcriptional and translational control elementscan therefore be provided between the T-DNA borders of a Ti-plasmid. Theplasmid may be a disarmed Ti-plasmid from which the genes fortumorigenicity have been deleted. The sul gene and its transcriptionaland control elements can, however, be provided between T-DNA borders ina binary vector in trans with a Ti-plasmid with a vir region. Such abinary vector therefore comprises:

(a) the chimaeric gene under the control of regulatory elements capableof enabling the gene to be expressed when integrated in the genome of aplant cell; and

(b) at least one DNA sequence which delineates the DNA to be integratedinto the plant genome.

Agrobacterium tumefaciens, therefore, containing a hybrid plasmid vectoror a binary vector in trans with a Ti-plasmid possessing a vir regioncan be used to transform plant cells. Tissue explants such as stems orleaf discs may be inoculated with the bacterium. Alternatively, thebacterium may be co-cultured with regenerating plant protoplasts. Plantprotoplasts may also be transformed by direct introduction of DNAfragments which encode the mutated DHPS and in which the appropriatetranscriptional and translational control elements are present or of avector incorporating such a fragment. Direct introduction may beachieved using electroporation, polyethylene glycol, microinjection orparticle bombardment.

Plant cells from monocotyledonous or dicotyledonous plants can betransformed according to the present invention. Monocotyledonous speciesinclude barley, wheat, maize and rice. Dicotyledonous species includetobacco, tomato, sunflower, petunia, cotton, sugarbeet, potato, lettuce,melon, soybean, canola (rapeseed) and poplars. Tissue cultures oftransformed plant cells are propagated to regenerate differentiatedtransformed whole plants. The transformed plant cells may be cultured ona suitable medium, preferably a selectable growth medium. Plants may beregenerated from the resulting callus. Transgenic plants are therebyobtained whose cells incorporate a sul gene in their genome, the sulgene being expressible in the cells. Seed from the regenerated plantscan be collected for future use.

The growth of weeds can be controlled by the use of a herbicide whichacts by inhibiting dihydropteroate synthase, such as asulam or anagriculturally acceptable salt thereof, at a locus where plantsaccording to the invention are under cultivation. As these plants areresistant to the herbicide due to expression of a mutated DHPS, theherbicide or a mixture of such herbicides can be applied without fear ofdamaging the plants. The herbicides may be applied pre- and/orpost-emergence of the weeds and/or the plants. Suitable agriculturallyacceptable salts include the alkali metal (particularly sodium andpotassium), ammonium, amine (particularly diethanolamine,triethanolamine, octylamine, morpholine and dioctylmethylamine) andalkaline earth metal (particularly calcium and magnesium) salts.

Herbicides which act by inhibiting dihydropteroate synthase, such asasulam and its salts, are active against monocotyledonous anddicotyledenous weeds. They exhibit a low toxicity to mammals, birds,fish and wild life in general, and have a short soil persistence. Theymay be used, for example at rates of application of from 1.0 to 4.0 kgasulam equivalent/hectare, by post-emergence application to control thegrowth of annual and perennial grass weeds such as:

Agropyron repens, Agrostis gigantea, Agrostis stolonifera, Alopecurusmyosuroides, Avena fatua, Brachiaria eruciformis, Bromus tactorum,Cenchrus pauciflorus, Cynodon dactylon, Digitaria sanguinalis, Digitariascalarum, Digitaria spp. Echinochloa colonum, Echinochloa crus-galli,Eleusine indicia (=africana), Eragrostis sp, Hordeum murinum, Holcuslanatus, Imperata cylindrica, Leptochloa filiformis, Panicumfasciculatum, Panicum purpurascens, Paspalum dilatatum, Poa annua, Poatrivialis, Rottboellia exaltate, Setaria spp and Sorghum halepense;

and annual and perennial broad-leafed weeds such as:

Ambrosia sp, Anthemis sp, Artemisia sp, Bellis perennis, Cirsiumarvense, Erigeron sp, Galinsoga parviflora, Matricaria spp, Seneciojacobaea, Sonchus oleraceus, Tussilago farfara, Capsella bursa-pastorisErysimum cheiranthoides, Raphanus raphanistrum, Sinapis arvensis,Polygonum aviculare, Polygonom convolvulus, Polygonum persicaria, Rumexcrispus, Rumex obtusifolius, Equisatum arvensis and Pteridium aquilinum.

The herbicides, in particular asulam and its agriculturally acceptablesalts, are normally applied as spray fluids. Such spray fluids may, ifdesired, contain from 0.1% to 1% w/v of surfactant. Suitable surfactantsmay be of the ionic or non-ionic types, for example sulphoricinoleates,quaternary ammonium derivatives, products based on condensates ofethylene oxide with nonyl- or octyl-phenols, or carboxylic acid estersof anhydro-sorbitols which have been rendered soluble by etherificationof the free hydroxy groups by condensation with ethylene oxide, alkaliand alkaline earth metal salts of sulphuric acid esters and sulphonicacids such as dinonyl- and di-octyl-sodium sulpho-succinates and alkaliand alkaline earth metal salts of high molecular weight sulphonic acidderivatives, e.g. sodium and calcium lignosulphates.

Such spray fluids may be obtained from concentrates comprising asulam oran agriculturally acceptable salt thereof. Compositions in the form ofconcentrates which are to be diluted, e.g. with water or oil, before useas herbicides may, for example, contain from 0.05% to 10% of surfactant,e.g. from 0.05% to 10% w/w of surfactant in the case of solidconcentrates and from 0.05% to 10% w/v of surfactant in the case ofliquid concentrates.

Herbicidal compositions containing the herbicide may also containdiluents or carriers and/or, if desired, conventional adjuvants such asadhesives, colouring agents and corrosion inhibitors. These adjuvantsmay also serve as carriers or diluents. Examples of suitable soliddiluents or carriers are water-soluble alkali metal (e.g. sodium orpotassium) chlorides (e.g. sodium chloride), carbonates, bicarbonatesand sulphates, and urea. The solid compositions, which may take the formof water soluble powders or granules, are preferably prepared bygrinding the active ingredient with solid diluents. Granularformulations may be prepared by granulating compositions in powder formobtained as described above.

Liquid compositions may take the form of aqueous solutions orsuspensions in oil which may incorporate surfactants. Suitable liquiddiluents for incorporation in the liquid compositions include water andaliphatic and aromatic hydrocarbons. Surfactants, which may be presentin the liquid compositions, may be of the type described above and may,when liquid, also serve as diluents or carriers. Liquid compositions inthe form of concentrates may be diluted with water or an oil, e.g.kerosene, to give compositions ready for use.

A sul gene can also be used as a selectable marker for celltransformations on the basis that cells transformed with the gene areresistant to a sulfonamide such as sulfadiazine, asulam or one of itssalts. This applies not only to plant cell transformations but also totransformations of other cells, for example bacteria. A sul gene can beco-introduced with a gene encoding a second trait into cells. The cellsare screened for resistance to a sulfonamide or salt thereof. Cellsresistant to the sulfonamide or sulfonamide salt are screened for thegene coding for the second trait.

This second trait may be another agriculturally desirable property suchas resistance to another herbicide. The second trait may be a trait forwhich it is difficult to assay. In the context of plants, the sul genemay therefore be linked to a gene coding for a storage protein such asphaseolin; a lectin; a resistance factor against a disease, an insect oranother herbicide; or a factor providing tolerance to environmentalstress. Any sul gene may be employed; although the gene is typically abacterial gene. The gene may be a sulI or sulII gene. A sul geneaccording to the invention may be provided, although optionally theremay be base changes which do or do not result in amino acidsubstitutions in the expressed product. A chimaeric gene as describedabove is used when the sul gene is being used as a selectable marker forplant cell transformations.

For plant cells, a chimaeric sul gene is provided between T-DNA borders.The gene encoding the second trait of interest is also provided betweenT-DNA borders. If the two genes are provided between the same T-DNAborders, preferably the chaimeric sul gene is closer to the left bordersequence. The gene encoding the second trait of interest is also in theform of a chimaeric gene comprising a plant promoter for the codingsequence and a plant polyadenylation/terminator sequence.

A gene encoding a second trait of interest can, therefore, be introducedwith a sul gene into cells whose growth is sensitive to inhibition by asulfonamide such as sulfadiazine, asulam or an agriculturally acceptablesalt thereof. A single DNA fragment containing both genes or two DNAfragments each containing a different one of the genes may be employed.The cells can then be grown in a medium containing the sulfonamide orsalt thereof. Cells which survive can be assumed to have beentransformed not only with the sul gene but also with the gene encodingthe second trait. The genes can be introduced into the cells to betransformed directly or by any appropriate vector, for example aplasmid, phage (for bacteria) or virus.

In the present invention, resistance or sensitivity of cells to asulfonamide such as sulfadiazine, asulam or one of its salts is assessedby the ability of the cell to grow, to grow less well or not to grow inthe presence of a predetermined concentration of the sulfonamide or saltthereof. This concentration may be selected depending uponcircumstances. It may be 10, 25, 50, 100, 250 or 500 μg, calculated asthe sulfonamide per ml of the medium in which the cells are present.That resistance of cells or regenerants is due to the integration andexpression of the sul gene may be verified by assaying for the presenceof sulfonamide-insensitive DHPS.

The following Examples illustrate the invention. In the accompanyingdrawings:

FIGS. 1(A and B) shows the sub-cloning of fragments into pUC vectors.Thick lines represent the sequences originating from plasmid R46, thinlines show the vectors. Small arrows indicate the direction oftranscription initiated by the lac promoter. Vectors (pBR322, pUC12,pUC19) were treated with alkaline phosphatase during their digestionwith restriction enzymes. T₄ DNA ligase was used for ligating a vectorand insert or for circularising deleted plasmids. MCS representsmultiple cloning sites.

FIG. 2 shows the growth of E. coli strain JM101 harbouring variousplasmids on minimal medium containing asulam at 500 μg/ml. Bacteria withno plasmid, dots; pUC12sul, closed triangles; pUC19sul, open triangles;and pBRsulΔ3, closed circles.

FIG. 3 shows the strategy for sequencing the fragment from pBRsulΔ3containing the sul gene. The arrows indicate the direction ofsequencing. The upper arrow shows the position of the coding sequence.Not all the AluI, AvaII, DdeI sites are indicated.

FIG. 4 shows the nucleotide sequence of the 1.8 kb fragment of plasmidR46 present in pBRsulΔ3. The first start codon of the open reading frame(ORF) and the stop codon are boxed. The putative ribosome-binding-siteis in an open box. The predicted amino-acid sequence of the mutated DHPSencoded by the sul gene is shown under the nucleotide sequence.

FIGS. 5(A and B) shows the cloning of the sul gene in frame with the lacpromoter into pUC19, giving pJIT92. At each step, T₄ DNA ligase was usedto ligate the fragments of DNA. Vectors were treated with alkalinephosphatase during their digestion with restriction enzymes. Thedownstream BsmI site in the sul gene includes the stop codon which iseliminated by the T₄ DNA polymerase treatment but restored when the BsmIfragment is inserted into a filled-in HindIII site. The nucleotidesequence in the vicinity of the start and stop codons of the sul gene inpJIT92 was checked by sequencing. The sequences indicate the translationframes in pJIT92 and pHIT92ΔNc. The four extra-nucleotides inducing theframe shift in pJIT92ΔNc are underlined.

FIG. 6 shows the growth of E. coli strain JM101 harbouring no plasmid,dots; pHJIT92, closed squares; and pJIT92ΔNc, open squares; on minimalmedium containing asulam at 500 μg/ml.

FIG. 7A shows the organization of pJIT119. A. 35S, CaMV 35S promotersequence; TP, rbc5 transit peptide sequence; C., CaMV sequencecontaining transcription terminator and polyadenylation sequence. FIG.7B. Upper lines: nucleotide and amino acid sequences of rbcS (Andersonand Smith, Biochem J. 240, 709-715, 1986). Arrow indicates the point ofcleavage between the transit peptide and the mature protein. Bottomlines: nucleotide and amino acid sequences at the junction between therbcS transit peptide sequence and the sul coding sequence in pJIT119.

EXAMPLE 1 Isolating and Examining the sul gene

(a) Cloning of a fragment of plasmid R46 carrying the sul gene

A 1.25 kB HindIII-Sstl fragment from within the 2.8 kb EcoR1-Sallfragment of plasmid R46 (Brown and Willette, plasmid, 5, 188-201, 1981)was cloned into pUC vectors (Yanish-Peron et al., Gene 33, 103-119,1985) as shown in FIG. 1. In brief, the 1.25 kb HindIII-SstI fragmentwas electroeluted from an agarose gel after cutting with HindIII andSstI. This fragment was ligated to pUC12 or pUC19 cut with the sameenzymes to create pUC12sul and pUC19sul respectively.

The ability of pUC12sul and pUC19sul to confer resistance to asulam wasassessed by growing E. coli JM101 harbouring either plasmid on minimalmedium containing 500 μg of asulam per ml. The results are shown in FIG.2. These show that the 1.25 kb HindIII-SstI fragment does not allowbacteria to grow on asulam at 500 μg/ml when cloned into theHindIII-SstI sites of pUC12 but that the fragment does allow bacteria togrow on asulam at 500 μg/ml when cloned into the same sites of pUC19.

These data indicate that the sul gene is orientated from the HindIIIsite towards the SstI site in the HindIII-BstI fragment and that thisfragment contained the coding sequence or an essential part of it, butnot all the information required for the expression of the gene. The lac5' sequence is thought to replace the equivalent sul sequence missing inpUC19sul.

Plasmid pBRsulΔ1 was obtained by deleting 550 bp from the EcoRI end ofthe 2.8 kb EcoRI-Sall fragment of R46 using BAL31 and by cloning theresulting fragment into pBR322 as shown in FIG. 1. The EcoRI-ClaIfragment from pBR322, containing the -35 sequence of the TC^(r)promoter, was deleted in pBKsulΔ1 to give pBRsulΔ2. A 300 bp SstI-Sallfragment was deleted at the other extremity of the R46 sequenceremaining in pBRsulΔ2, giving pBRsulΔ3. The growth of E. coli JM101harbouring pBKsulΔ3 in minimal medium containing asulam at 500 μg/ml isshown in FIG. 2. This indicates that none of the deletions affect theresistance to asulam. Consequently, the 1.8 kb sequence remaining inpBRsulΔ3 contains all the information required for the expression of thesul gene.

(b) Nucleotide sequencing

The strategy used to determine the nucleotide sequence of the 1.8 kbfragment from R46 remaining in pBRsulΔ3 is shown in FIG. 3. The sequencewas determined using the dideoxy chain extension method (Sanger et al.,Proc. Natl. Acad. Sci. USA 74, 5463-5467, 1977) as described byMullineaux et al., (EMBO J., 3, 3063-3068, 1984), except that [³⁵ S]daTPwas used for radiolabelling. In some cases, dITP and Sequenase (UnitedStates Biochemical Corp.) were used in the reactions instead of dGTP andKlenow polymerase. The sequence is presented in FIG. 4. An open readingframe (ORF) of 840 bp, preceded by a putative Shine and Delgarnosequence, was present in the sequence.

(c) Expression of the sul gene

The putative coding sequence was recovered from pUC19sul and placed inframe with the first codons of the lacZ gene in pUC19, to form pJIT92 asshown in FIG. 5. This was achieved as follows. pUC19sul was cut withStyI. After treatment with T₄ polymerase to fill in the ends, the smallfragment containing the sul start codon was eluted from an agarose geland cloned into the HindIII site of pUC19, giving pUCsul(Sty), in whichthe lacZ ATG and the sul ATG are in frame. The BsmI fragment of pUC19sulwas eluted from an agarose gel after treatment with T₄ polymerase andcloned into the filled-in HindIII site of pUC9, giving pUCsul(Bsm).

The sul stop codon present in the BsmI site is thus restored by theHindIII (confirmed by sequencing). To reconstruct the entire sul gene,the SstII-BamHI fragment from pUCsul(Bsm) was inserted into the samesites of pUCsul(sty), creating pJIT92, which contains the entire sulcoding sequence in frame with the lacZ first codons. E. coli JM101harbouring pJIT92 were deposited at the National Collection ofIndustrial and Marine Bacteria, Aberdeen, GB on 26 Oct. 1989 underaccession number NCIMB 40218.

A frame shift was created between the lacZ ATG and the sul ATG, creatingpJIT92ΔNc. This was achieved by cutting pJIT92 with NccI, followed bytreatment with T₄ polymerase and self-ligation so that four bases wereadded between the lacZ and sul start codons. The frame shift is shown inFIG. 5.

Asulam resistance was determined as in (a) above. The results are shownin FIG. 6. These indicate that the expression of the sul gene was drivenby the lac promoter in pJIT92. Translation of the mRNA would lead to thesynthesis of the predicted protein when initiated at the sul ATG and ofa fusion protein when initiated at the lacZ ATG.

The frame shift present in pJIT92ΔNc reduced the level of resistance toasulam to a level close to the one induced by pUC19sul. The frame shiftwould have resulted in the synthesis of an abnormal protein when thetranslation was initiated at the lacZ ATG. In some cases, thetranslation may have been initiated at the sul ATG, generating a certainamount of functional protein. This would explain the residual resistanceto asulam of bacteria having pJIT91ΔNc as seen in FIG. 6. These resultsconfirm that the ORF recovered from pUC19sul coded for sulphonamideresistance.

(d) Analysis of the protein sequence

The amino acid sequence of the mutated DHPS was dedeuced from thenucleotide sequence shown in FIG. 4. The predicted protein contained 279amino acids and its molcular weight is 30106. The protein possesses ahydrophobic --NH₂ end and a hydrophilic --COOH end. The amino acidsequence showed no significant homology with the 4931 sequences presentin the PIR data base (release 15.0) of the National Biomedical ResearchFoundation (Aberdeen, GB).

EXAMPLE 2 Plant Transformation

1. Material and methods

(a) Molecular cloning

In a first step to constructing pJIT119, the sul coding sequence wasrecovered from pJIT92 and inserted into the polylinker of pJIT117(Guerineau et al., Nucleic Acids Res. 16, 11380, 1988). pJIT92 was cutwith NcoI, treated with T₄ polymerase, cut with SalI and the digestedfragments were separated by electrophoresis in an agarose gel. Thefragment containing the sul gene was eluted from the gel as described inManiatis et al. (Molecular Cloning--A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York, 1982) and ligated to pJIT117previously cut with SphI, treated with T₄ polymerase and cut with SalI,creating pJIT118. The nucleotide sequence at the cloning junction waschecked after subcloning a HindIII-PstI fragment carrying the junctioninto M13mp18 (Yanisch-Perron et al., 1985). The nucleotide sequence wasdetermined by the dideoxy-chain extension method (Sanger et al., 1977).

pJIT58 was constructed by insertion of the gus coding sequence recoveredfrom pBI101-2 (Jefferson et al., EMBO J. 6 3901-3907, 1987), intopJIT30. pJIT30 is an expression cassette made of a pUC-derived vectorcarrying the sequence from CaMV Cabb-JI corresponding to coordinates7040-7432 (Franck et al., Cell 21, 285-294, 1980) and containing the 35Spromoter, a polylinker and the sequence 7435 to 126 from CaMV providinga polyadenylation signal. The fusion 35S-gus-poly A was moved frompJIT58 into the polylinker of pBIN19 (Bevan, Nucleic Acids Res. 12,8711-8721 1984) as a KpnI-XhoI fragment, generating pJIT59.

The fusion 35S promoter-rbcS transit peptide (TP)-sul-polyadenylationsequence was recovered from pJIT118 as a KpnI fragment and cloned intothe KpnI site of pJIT59, upstream from the gus gene, giving pJIT119. Theorganisation of pJIT119 is shown in FIG. 7. pGEMTPSUL was constructed byinsertion of the fusion transit peptide-sul recovered from pJIT118 as aHindIII-EcoRI fragment, into the polylinker to pGEM-4Z (Promega). E.coli JM101 harbouring pJIT119 was deposited at the National Collectionof Industrial and Marine Bacteria, Aberdeen GB on 26 Oct. 1989 underaccession number NCIMB 40219.

(b) Chloroplast import experiments

The sul and TP-sul genes cloned into pGEM-4Z were transcribed under SP6RNA polymerase, and capped transcripts were translated in a wheat-germlysate in the presence of [³⁵ S]-methionine (Anderson et al., MethodsEnzymol. 101, 635-644, 1983; Malton et al., Nucleic Acids Res. 12,7035-7056, 1984). The translation products were incubated with isolatedpea leaf chloroplasts as described (Robinson and Ellis, Eur. J. Biochem.152, 67-73, 1985). After import, the chloroplasts were incubated withtrypsin to digest non-imported proteins, and the organelles werefractionated into stromal and membrane samples. Samples were analysed bysodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresisfollowed by fluorography.

(c) Plant transformation

Nicotiana tabacum cv Samsun plants were grown in a glasshouse understandard conditions. Approximately 1 cm² leaf pieces were cut fromsurface sterilized leaves and floated on MS medium (Murashige and Skoog,Physiol. Plant 15, 473-497, 1962) containing 3% sucrose, 1 mg/l6-benzylaminopurine (BAF), 0.1 mg/l α-naphthaleneacetic acid (NAA) and 3mM 2-[N-morpholino]-ethanesulfonic acid (NES), pH 5.7. Bacteria obtainedfrom overnight cultures of Agrobacterium tumefaciens strain LBA4404(Hoekema et al., Nature 303, 179-180, 1983) containing pJIT59 orpJIT119, were centrifuged and resuspended in the same volume of MSmedium. One teeth of volume was added to the floating leaf pieces.

Cocultivation was carried out for three days at 25° C. under light(Photoperiod 16 h/8 h). Leaf pieces were blotted dried onto absorbingpaper and placed on MS medium as above but containing 0.8% agar, 500μg/ml of ampicillin, 250 μg/ml cefotaxime and the selective agent atvarious concentrations. Asulam or sulfadiarine was used for selectingtransfrmants.

After one month culture, shoots, developed on leaf pieces placed onselective media, were cut off and rooted on 0.5×MS medium containing 1%sucrose, 1% agar, 3 mM MES pH 5.7, 200 μg/ml amplicillin, 100 μg/mlcefotaxime and eventually sulfadiazine as indicated. Plantlets weretransferred to soil when rooted, generally after a month, and grownuntil seeds could be harvested. Seeds were sterilized in 10% (vol)Domestos (Trade Mark) for 15 min and washed four times in steriledistilled water. Germination took place in Petri diskes containing0.5×MS medium supplemented with 1% sucrose, 1% agar and sulfadiazine asindicated in the text.

(d) Plant DNA extraction and analysis

Plantlets were ground in 2 ml of grinding buffer (Tris 100 mM pH 8,ethylene diamine tetracetic acid (EDTA) 20 mM, SDS 0.1%,diethyldithiocarbamate 0.5%) per g fresh weight of tissue. Twoextractions with one volume of water-saturated phenol/chloroform (1vol/1 vol) were carried out and nucleic acids were ethanol precipitated.Pellets were dissolved in one ml/g of distilled water and one volume ofa solution containing 13% polyethyleneglycol (PEG) 6000 and 0.8M NaClwas added to the aqueous phase. Precipitate was allowed for 30 min at 0°C. DNA was recovered by centrifugation for 5 min at 10000 g. Pelletswere washed with 70% ethanol, dried and dissolved in distilled water.DNA concentration was determined using the fluorimetric method asdescribed by Thomas and Farquhar (Anal. Biochem. 89, 35-44, 1978).

For analysis, 10 μg of DNA was digested overnight with 100 units ofEcoRI and electrophoresed through a 0.8% agarose gel. The gel wasprocessed and DNA was blotted onto nitrocellulose membrance as describedin Maniatis et al. (1982). Prehybridization was carried out for 3 h at65° C. in 5× SSC (1× SSC is 15 mM sodium citrate, 150 mM NaCl), 25 mMsodium phosphate buffer pH 6.5, 0.1% SDS, 5 mM EDTA, 5× Denhart solution(1× Denhart solution is 0.02% Ficoll, 0.02% polyvinylpyrrolidone (PVP),0.02% bovine serum albumin (BSA)), 100 μg/ml denatured calf thymus DNA.35 ng of sul DNa fragment was labelled with [³² P]dCTP using the randomoligonucleotide priming method (Feinberg and Vogelstein, Anal. Biochem.132, 6-13, 1983) to a specific activity higher than 10⁵ counts perminute (CPM)/μg. Hybridization was carried out overnight at 65° C. inthe prehybridization solution. The membrane was washed in 2× SSC, 0.1%SDS for 30 min at room temperature, then in 2× SSC, 0.1% SDS for 30 minat 65° C., then twice in 0.1× salt and sodium citrate (SSC), 0.1% SDS,for 30 min at 65° C. The membrane was exposed to preflashed X-ray filmfor 2 days.

2. Results

(a) Construction of pJIT119 containing a sul chimeric gene

In order to target the bacterial DHPS to chloroplasts, the sul codingsequence was fused to the pea ribulose bis-phosphatecarboxylase/oxygenase (rbcS) transit peptide sequence contained in theexpression cassette pJIT117 (Guerineau et al., 1988). The fusion wasmade so that the sul ATG translation initiation codon was exactly in thesame position as the mature rbcS ATG in the native enzyme (FIG. 7). Thebacterial DHPS was expected to be released from the transit peptide inthe chloroplast stroma, in the same form as it is synthesized inbacterial, i.e. not as a fusion protein. For transcription of thechimeric gene, a CaMV 35S promoter with a duplicated enhancer region andCaMV gene VI polyadenylation signal were placed at the 5' and 3' endsrespectively of the TP-sul coding sequence. In the final construct usedfor plant transformation, the chimeric sul gene was inserted between theright and the left T-DNA border of the binary vector pBIN19 (Bevan,1964) which also carried the chimaeric uida gene (β-glucuronidase (gus))gene (Jefferson et al., 1966) placed under control of the 35S promoter(FIG. 7). The construct was mobilized into Agrobacterium tumefaciensdisarmed strain LBλ4404 (Hoekman et al., 1983) using the helper plasmidpRK2013.

(b) In vitro transport of DMPS into isolated chloroplasts

In order to confirm that the rbcS transit peptide is capable oftargetting DHPS into chloroplasts, we tested whether chloroplasts arecapable of importing the fusion protein (in vitro). The portein (TPDHPS) was synthesized by the in vitro transcription followed bytranslation in a wehat-germ lysate. This expression system generatedTP-DHPS and also a protein of lower M_(r). The latter corresponds tomature-sized DHPS which is probably generated by internal initiation; apolypeptide of identical M_(r) was synthesized if DHPS cDNA is expressedby this method.

TF-DHPS was imported into the stroma of isolated pea chloroplasts andprocessed to a lower M_(r) form. The processed protein had a mobilitysimilar to that of mature-sized DHPS, suggesting that the entirepre-sequence had been removed. In control experiments (not shown), itwas found that mature-sized DHPS (lacking the transit peptide sequence)was not important by chloroplasts under these conditions. These datashow that the rbcS RUBISCO transit peptide sequence effectively targetsDHPS into the chloroplast stroma.

(c) Transformation of tobacco leaf explants with pJIT119

During preliminary experiments, our sul gene was placed under control ofthe CaMV 35S promoter and transferred to tobacco plants. Althoughevidence was obtained that the gene was transcribed, no increase in thelevel of resistance to sulfonamides was recorded in transformed leaftissues (data not shown).

Tobacco leaf pieces were then cocultivated with Agrobacteriumtumefaciens strain LBA4404 carrying pJIT119, and placed on mediumcontaining 50 μg/ml of kanamycin. No shoots developed on untreatedcontrol laser pieces whereas a number of shoots grew on cocultivatedleaf pieces. Shoots were rooted and assayed for β-glucuronidase (GUS)activity. Leaf pieces from four of the plantlets showing GUS activityand from an untransformed control were placed on medium containingvarious concentrations of sulfadiazine.

No shoots developed on untransformed control leaf pieces after threeweeks on sulfadiazine at 5 μg/ml, whereas shoots developed on leafpieces prepared from three of the plantlets showing GUS activity, onmedium containing up to 500 μg/ml of sulfadiazine. These data indicatethat a high level of resistance to sulfadiazine was present in leaftissue of plantlets displaying GUS activity after cocultivation withAgrobacterium tumefaciens carrying pJIT119.

(d) Direct selection of transformants or sulfonamides

To determine if the level of resistance to sulfadiazine recorded inshoots selected on kanamycin was sufficient to allow direct selection oftransformants, tobacco leaf explants were cocultivated withAgrobacterium tumefaciens carrying pJIT119 and placed on mediumcontaining asulam or sulfadiazine at 100 μg/ml. This value was chosenbecause untransformed leaf explants did not produce any shoots for onemonth, on medium containing that concentration.

In a first experiment, asulam was used as the sole selective agent.Shoots developed on leaf explants cocultivated with Agrobacteriumharbouring pJIT119 but not on those treated with bacteria carryingpJIT59, a pBIN19 derivative carrying the gus gene but not having the sulgene. Seven shoots growing on leaf explants cocultivated withAgrobacterium carrying pJIT119 were rooted and plants were regenerated.DNA was extracted from plantlets grown from seeds of each of theselected plants and probed for the presence of the bacterial sul gene.Progeny of all seven plants was shown to contain the bacterial sequence,indicating that the shoots selected on asulam were transformants.

In a second experiment, sulfadiazine was used as the sole selectiveagent. Nine shoots selected on leaf explants cocultivated withAgrobacterium harbouring pJIT119 were placed on rooting mediumcontaining sulfadiazine at 100 μg/ml. Eight rooted and grew normally andone did not. All shoots showed specific GUS activity higher than 100times the background activity of untransformed material. None of twelveuntransformed control shoots rooted and their growth was completelyinhibited on sulfadiazine at the same concentration.

In a third experiment, fifteen shoots selected on asulam were placed onrooting medium containing 100 μmg/ml of sulfadiazine. Three did notproduce any roots, did not grow and turned yellow, one grew very slowlyand produced only one root, eleven grew and rooted normally. GUS assayson rooted shoots showed that ten out of twelve had high GUS activity.Leaf pieces from the two rooted plantlets not showing any GUS activitywere challanged on sulfadiazine at 100 μmg/ml. They both produced shootsnormally whereas untransformed controls bleeched, suggesting that thesul gene was expressed in those two selected shoots although the gusgene was not. These three experiments demonstrated that the directselection on sulfonamides, of transformed shoots on leaf explants aftertransformation with pJIT119, was efficient.

(e) Analysis of the progency of transformed plants

Seeds harvested from two self-fertilized transformed plants during thefirst selection experiment and seeds from an untransformed controlplant, were germinated on medium containing various concentrations ofsulfadiazine. Seedlings from the untransformed seeds died soon aftergermination, 20 μg/ml of sulfadiazine and more, whereas transformedseedlings developed normally on sulfadiazine at concentrations up to 200μg/ml. The growth of transformed seedlings was slightly reduced onsulfadiazine at 500 μg/ml.

In order to record the pattern of segregation of the introducedcharacter, seeds from the seven transformed plants obtained during thefirst selection experiment were germinated on sulfadiazine at 100 μg/ml.Growth was monitored for two weeks. Seedlings developed normally fromthe seed harvested on 5 out of 7 transformed plants. The segregation ofsulfonamide resistance is shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                 Number of seeds                                                      Plant   resistant      sensitive                                                                              Ratio                                         ______________________________________                                        1       117            38       3.1/1                                         2       120            34       3.5/1                                         3       76             24       3.2/1                                         4       77             23       3.2/1                                         5       68             32       2.1/1                                         ______________________________________                                    

The segregation patterns were in most cases three to one which is to beexpected from a dominant character encoded by a determinant integratedinto the genome at one locus. No difference in the growth of seedlingson sulfadiazine, indicating homozygotic or heterozygotic status, even at500 μg/ml, could be observed. In contrast, GUS assays allowed a cleardistinction between seedlings having one or two alleles of the insertedT-DNA (data not shown). No seedling grew on sulfadiazine at 100 μg/ml,from the seed harvested on two out of the seven transformed plants.However, in both cases, approximately 75% of the seedlings had formedyellow or even green cotyledons, after one month on media containingsulfadiazine at 100 μg/ml. A very low GUS activity, althoughsignificantly higher than the activity of untransformed controlseedlings, was detected in some of the seedlings. This seemed toindicate that the sul and the gus genes were only very weakly expressedin the progency of these two plants.

Overall, these data indicate that the sulfonamide resistance gene wastransmitted to the progency of transformed plants and was expressed as adominant character.

We claim:
 1. A plant cell whose growth is resistant to inhibition by asulfonamide or a salt thereof, which plant cell has been obtained by:(i)transforming a plant cell whose growth is sensitive to inhibition bysaid sulfonamide or a salt thereof with a chimaeric gene comprising (a)a plant promoter, (b) a a sul gene which encodes a modifieddihydropteroate (DHPS) conferring sulfonamide resistance, or a sequencehaving at least 70% homology thereto conferring sulfonamide resistanceand which has a sequence encoding a transit peptide cleavable from themodified DHPS fused to the 5'-end of the resistance gene and (c) a plantpolyadenylation/terminator sequence wherein (a), (b) and (c) areoperably linked; and (ii) selecting a transformed plant cell whosegrowth is resistant to inhibition by said sulfonamide or salt thereof.2. A cell according to claim 1, wherein the promoter of the chimaericgene is selected from the 35S cauliflower mosaic virus promoter or anopaline synthase promoter or octopine synthase promoter.
 3. A cellaccording to claim 1, wherein the chimaeric gene encodes a modified DHPShaving the amino acid sequence; ##STR3## or at least 70% homology tosaid sequence provided that resistance to a said sulfonamide isconferred on the cell when the said gene is expressed therein.
 4. A cellaccording to claim 3, wherein the said gene has the DNA sequence;##STR4## or at least 70% homology to said sequence modified by one ormore codon insertions and/or deletions provided that resistance to asaid sulfonamide is conferred on the cell when the said gene isexpressed therein.
 5. A cell according to claim 1, wherein the transitpeptide sequence of the chimaeric gene encodes the transit peptide forribulose-1,5-bisphosphate carboxylase/oxygenase.
 6. A cell according toclaim 1, wherein the said sulfonamide is asulam.
 7. A transgenic plantexhibiting resistance to a sulfonamide or a salt thereof, which plantcontains in its cells a chimaeric gene comprising (a) a plant promoter(b) a sul gene which encodes a modified DHPS conferring sulfonamideresistance, or a sequence having at least 70% homology theretoconferring sulfonamide resistance and which has a sequence encoding atransit peptide cleavable from the modified DHPS fused to the 5'-end ofthe resistance gene and (c) a plant polyadenylation/terminator sequencewherein (a), (b) and (c) are operably linked.
 8. A plant according toclaim 7 wherein the promoter of the chimaeric gene is selected from the35S cauliflower mosaic virus promoter or a nopaline synthase promoter oroctopine synthase promoter.
 9. A plant according to claim 7 wherein thechimaeric gene encodes a modified DHPS having the amino acid sequence;##STR5## or at least 70% homology to said sequence provided thatresistance to a said sulfonamide is conferred on the plant when the saidgene is expressed therein.
 10. A plant according to claim 9 wherein thesaid gene has the DNA sequence; ##STR6## or at least 70% homology tosaid sequence provided that resistance to a said sulfonamide isconferred on the plant when the said gene is expressed therein.
 11. Aplant according to claim 7 wherein the transit peptide sequence of thechimaeric gene encodes the transit peptide for ribulose-1,5-bisphosphatecarboxylase/oxygenase.
 12. A plant according to claim 7 wherein the saidsulfonamide is asulam.
 13. Seed obtained from a transgenic plant asclaimed in claim
 7. 14. A method of controlling the growth of weeds at alocus where a transgenic plant as claimed in claim 7 is beingcultivated, which method comprises applying to the locus an effectiveamount of a sulfonamide or salt thereof, as herbicide, which acts byinhibiting dihydropteroate synthase.
 15. A method of treating a locus inwhich transgenic plants as claimed in claim 7 are being cultivated,which comprises applying to the locus an effective amount of asulfonamide or a salt thereof, as herbicide, which acts by inhibitingdihydropteroate synthase.
 16. A plant cell according to claim 1, whereinsaid plant cell is a dicotyledonous plant cell.
 17. A plant cellaccording to claim 1, wherein said plant cell is a monocotyledonousplant cell.
 18. A transgenic plant according to claim 7, wherein saidtransgenic plant is a dicotyledounous plant.
 19. A transgenic plantaccording to claim 7, wherein said transgenic plant is amonocotyledonous plant.
 20. A transgenic tobacco plant exhibitingresistance to a sulfonamide or a salt thereof which tobacco plantcontains in its cells a chimaeric gene comprising (a) a plant promoter(b) a sul gene which encodes a modified DHPS conferring sulfonamideresistance or a sequence having at least 70% homology thereto, and whichhas a sequence encoding a transit peptide cleavable from the modifiedDHPS fused to the 5'-end of the resistance gene and (c) a plantpolyadenylation/terminator sequence, wherein (a), (b) and (c) areoperably linked.
 21. A transgenic oilseed rape plane exhibitingresistance to a sulfonamide or a salt thereof which oilseed rape plantcontains in its cells a chimaeric gene comprising (a) a plant promoter(b) a sul gene which encodes a modified DHPS conferring sulfonamideresistance or a sequence having at least 70% homology thereto, and whichhas a sequence encoding a transit peptide cleavable from the modifiedDHPS fused to the 5'-end of the resistance gene and (c) a plantpolyadenylation/terminator sequence, wherein (a), (b) and (c) areoperably linked.